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doi: 10.1242/10.1242/dev.00166
1 Nina Ireland Laboratory of Developmental Neurobiology, Department of
Psychiatry, University of California San Francisco, San Francisco, CA
94143-0984, USA
2 Gene Center and Institute of Biochemistry, University of Munich, Feodor
Lynenstrasse 25, 81377 Munich, Germany
* Author for correspondence (e-mail: jlrr{at}cgl.ucsf.edu)
Accepted 11 September 2002
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Topography, Thalamocortical axons, Internal capsule, Neocortex, Basal ganglia, Dlx, Ebf1, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Levitt et al., 1997;
Monuki and Walsh, 2001;
Pallas, 2001;
Ragsdale and Grove, 2001;
Ruiz i Altaba et al., 2001;
Sur and Leamey, 2001;
O'Leary and Nakagawa, 2002).
During development, thalamocortical and corticothalamic axons grow into the
subcortical telencephalon, where they meet and continue on their paths to the
cortex and thalamus, respectively (Miller
et al., 1993; Metin and
Godement, 1996; Molnar et al.,
1998a; Auladell et al.,
2000). In this process, thalamic axons of a given nucleus grow
through the ventral thalamus, telencephalic stalk and basal ganglia to the
specific presumptive neocortical areas that they will invade
(Crandall and Caviness, 1984;
Catalano et al., 1991;
Kageyama and Robertson, 1993;
Miller et al., 1993;
Metin and Godement, 1996;
Molnar et al., 1998a;
Auladell et al., 2000). This
first level of topographic organization (an inter-areal map), in which a given
thalamic nucleus projects to a specific neocortical region, is important to
relay visual, auditory, somatosensory and motor information to different
neocortical areas. Within each neocortical area, thalamic axons establish
connections that form an intra-areal topographic map, creating a second level
of topographic organization (Schlaggar and
O'Leary, 1994; Agmon et al.,
1995). For example, the visual thalamus (dorsal lateral geniculate
nucleus, dLGN) specifically projects to the occipital neocortex (visual
cortical area), and within this region, a topographic map of visual space is
generated through the distribution of axonal terminals
(Kageyama and Robertson, 1993;
O'Leary et al., 1994;
Sur and Leamey, 2001).
The development of cortical-thalamic interconnections requires several
pathfinding steps. For example, structures in the embryonic diencephalon and
basal ganglia are implicated in providing signals that guide the growing
axons, through attractants (Metin and
Godement, 1996; Metin et al.,
1997; Richards et al.,
1997; Bagnard et al.,
1998; Braisted et al.,
1999; Braisted et al.,
2000), repellents (Bagnard et
al., 1998; Braisted et al.,
1999; Bagri et al.,
2002), or by producing pioneering axons
(Mitrofanis and Guillery,
1993). In particular, groups of cells in the basal ganglia have
been proposed to be intermediate targets for cortical and thalamic axons, by
producing attractants or forming transient projections to the thalamus
(Mitrofanis and Guillery,
1993; Metin and Godement,
1996; Tuttle et al.,
1999). In addition, it was shown that preplate and subplate
cortical axons, which pioneer the corticofugal pathway, form a scaffold that
guides thalamic axons into the neocortex
(McConnell et al., 1989;
Ghosh et al., 1990;
Ghosh and Shatz, 1992;
Ghosh and Shatz, 1993;
Molnar et al., 1998b;
McQuillen et al., 2002). These
observations provided the basis of the `handshake' hypothesis, which proposes
that thalamic and cortical axons require each other to reach to their targets
(Molnar and Blakemore, 1995;
Molnar et al., 1998a). Support
for this model comes from analysis of the Tbr1 and COUP-TFI
mutants, in which subplate defects impair the capacity of thalamocortical
axons to reach the neocortex (Zhou et al.,
1999; Hevner et al.,
2001; Hevner et al.,
2002). Similar results are also observed in Emx1/Emx2
mutants (K. M. Bishop, S. G., Y. Nakagawa, J. L. R. R. and D. M. M. O'Leary,
unpublished). Conversely, in Gbx2 mutant mice, which have major
thalamic defects, corticothalamic projections do not reach their target
(Miyashita-Lin et al., 1999;
Hevner et al., 2002). Taken
together, these studies provide insights into how thalamic and cortical axons
reach their respective target structure. However, very little is known about
the mechanisms that control the targeting of thalamic axons to specific
neocortical domains.
The chemoaffinity hypothesis stipulates that topographic projections are
generated through the expression of molecules that mediate repulsion and/or
attraction between the projecting axons and their target
(Sperry, 1963;
Goodhill and Richards, 1999).
This mechanism was shown to play a key role in the establishment of
retinotectal projections through Eph and ephrin protein interactions
(Drescher et al., 1997;
Goodhill and Richards, 1999;
Feldheim et al., 2000) and is
the prevailing model for the formation of topographic projections in the
nervous system. It is implicated in the targeting of reticulogeniculate axons
(Feldheim et al., 1998), of
limbic thalamic axons to the limbic cortex
(Barbe and Levitt, 1992;
Mann et al., 1998) and
hippocampal neurons to the septum (Gao et
al., 1996), as well as in the formation of a topographic map
within a neocortical area (Vanderhaeghen
et al., 2000). Recent studies show that several genes, including
genes encoding Eph/Ephrins, are expressed locally or in gradients in the
neocortex before and shortly after the arrival of thalamic inputs
(Bulfone et al., 1995;
Gao et al., 1998;
Nothias et al., 1998;
Donoghue and Rakic, 1999;
Mackarehtschian et al., 1999;
Miyashita-Lin et al., 1999;
Nakagawa et al., 1999;
Rubenstein et al., 1999;
Liu et al., 2000;
Sestan et al., 2001),
suggesting that the expression of localized cues within the neocortex may
control the targeting of thalamic axons. Consistent with this model, the
inactivation of Emx2 and COUP-TFI transcription factor
genes, that are expressed in high-caudal-low-rostral gradients in the cortical
primordium, induces a change in neocortical molecular regionalization as well
as a corresponding change in the pattern of thalamocortical connectivity
(Bishop et al., 2000;
Mallamaci et al., 2000b;
Zhou et al., 2001;
Muzio et al., 2002).
However, there is evidence that mechanisms operating outside of the
neocortex may participate in regulating the development of thalamocortical
topography. Explant culture experiments indicate that thalamic axons can
innervate any region of the neocortex in vitro, suggesting the absence of an
instructive code within the cortex (Molnar
and Blakemore, 1991; Molnar
and Blakemore, 1995). Based on a variety of studies, it has been
proposed that molecular and/or temporal interactions between cortical and
thalamic axons inside the internal capsule may regulate the regional
specificity of thalamocortical connections
(Molnar and Blakemore, 1991;
Ghosh and Shatz, 1992;
Ghosh and Shatz, 1993;
Molnar and Blakemore, 1995;
Molnar et al., 1998a).
So far, little is known about the mechanisms that control the initial
targeting of thalamic axons to specific neocortical domains and the proposed
models remain to be tested. To address this issue specifically, we searched
for mouse mutants that exhibited projection defects in subpopulations of
thalamic axons. We chose to analyze Ebf1 mutant embryos because they
have internal capsule pathfinding defects
(Garel et al., 1999).
Ebf1 (also known as Olf-1, O/E-1, COE1) encodes a
HLH transcription factor (Hagman et al.,
1993; Wang and Reed,
1993; Dubois and Vincent,
2001), and its inactivation affects basal ganglia development
(Garel et al., 1999). We show
that, in Ebf1-/- mutant embryos, axons from the dLGN are
misrouted inside the basal ganglia and the projections that do form between
thalamic nuclei and neocortical domains have a shifted topography. This shift
occurs in the absence of an apparent change in thalamic or neocortical
regionalization and is preceded by a shift in the positions of thalamic axons
in the basal ganglia. These results indicate that thalamic projections from
different nuclei are not formed independently of one another and raise the
possibility that defects in the basal ganglia of Ebf1-/-
embryos participate in shifting the early topography of thalamocortical axons.
To test whether defects in structures located along the path of thalamic axons
might shift thalamocortical topography, we analyzed Dlx1/2 mutants
that have defects in basal ganglia development
(Anderson et al., 1997).
Dlx1 and Dlx2 encode homeodomain transcription factors and
are not expressed in cortical projection neurons or in the dorsal thalamus
(Bulfone et al., 1993;
Stühmer et al., 2002). In
Dlx1/2-/- embryos, some thalamic axons fail to grow past
the basal ganglia and, as in Ebf1 mutants, thalamic axons that do
reach the neocortex have a shifted topographic organization in the neocortex
and basal ganglia. Taken together, our study suggests that the early
topography of thalamocortical projections is not strictly and solely governed
by information located within the neocortex and dorsal thalamus and that the
positioning of thalamic axons within the basal ganglia may have an important
role in organizing these projections.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were maintained in a C57/B16 background and
crossed to produce homozygous embryos. Dlx1/2 heterozygous mice
(Qiu et al., 1997) were
maintained in mixed 129G and C57/B16 genetic background, and crossed to
produce homozygous embryos. PCR genotyping of both lines was performed as
described previously (Anderson et al.,
1997; Qiu et al.,
1997; Garel et al.,
1999). Heterozygous embryos did not show any phenotype and were
used as controls. For staging of embryos, midday of the day of vaginal plug
formation was considered as embryonic day 0.5 (E0.5).
In situ hybridization
Embryos were fixed overnight in 4% paraformaldehyde (PEA) at 4°C. In
situ hybridization were performed on 80-100 µm thick vibratome sections as
described previously (Garel et al.,
1999) with the following probes: cadherin 6 (Cdh6) (a
gift of M. Takeichi); Cdh8 (a gift of M. Takeichi); COUP-TFI
(a gift of M. Tsai); Ebf1 (a gift of R. Grosschedl); Emx2 (a
gift of A. Simeone); Epha4 (a gift of A. Nieto); Epha7 (a
gift of A. Wanaka); ephrin A2 (Efna2 — Mouse Genome
Informatics; a gift of J. Flanagan); ephrin A5 (Efna5 — Mouse
Genome Informatics; a gift of U. Drescher); Fgfr3 (a gift of D.
Ornitz); Gbx2 (a gift of G. Martin); Id2 (Idb2
— Mouse Genome Informatics; a gift of M. Israel); Lhx9 (a gift
of S. Retaux); netrin 1 (Ntn1 — Mouse Genome Informatics; a
gift of M. Tessier-Lavigne); Sema6a (a gift of W. Snider). Sections
were mounted in glycerol and analyzed under a dissection microscope.
Axonal tracing
After overnight fixation in 4% PFA at 4°C, single crystals of the
fluorescent carbocyanide dye DiI (1,1'-dioctadecyl
3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular
Probes) or DiA (4-[4-(dihexadecyl amino)styryl]N-methyl-pyridinium
iodide; Molecular Probes) were placed in single or multiple locations in the
neocortex or dorsal thalamus (Godement et
al., 1987; Metin and Godement,
1996). After 4-7 weeks at room temperature in 4% PFA to allow dye
diffusion, the sample were embedded in 5% agarose and cut into 100 µm thick
sections on a vibratome. Counterstaining was performed using Hoechst (Aldrich
Chemicals) or SYTOX green (Molecular Probes) and digital images were taken
using a Spot II camera on a fluorescent microscope or dissection
microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
further characterize this phenotype, we performed axonal tracing experiments
using DiI injections. Broad injections in the lateral part of the dorsal
thalamus in E16 Ebf1-/- embryos showed that some thalamic
axons are misrouted into the amygdalar region
(Fig. 1A,B). These axons form a
thick tract that ends in this region, except for a few axons that grow
dorsally in the direction of the neocortex. On the contrary, at more rostral
levels, thalamic axons appear normal within the internal capsule and grow into
the neocortical intermediate zone (Fig.
1C,D). To identify the region of the thalamus that sends the
misrouted axons, we placed a DiI crystal in the amygdalar region in E16.5
Ebf1-/- embryos. Retrogradely labeled cells were
specifically found in a lateral thalamic area that has the characteristic
shape and position of the presumptive dorsal lateral geniculate nucleus (dLGN)
(Jones, 1985)
(Fig. 1E,F). Thus, in the
absence of Edf1, dLGN axons are misrouted in the amygdalar region,
whereas the rest of the thalamic axons normally grow into the internal capsule
and the neocortex.
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The topography of connections between the dorsal thalamus and the
neocortex is shifted in Ebf1-/- embryos
Axons from specific thalamic nuclei normally project towards particular
neocortical domains (Crandall and
Caviness, 1984; O'Leary et
al., 1994; Sur and Leamey,
2001). We thus investigated in Ebf1 mutants if dLGN axons
reach their final target, the occipital neocortex and whether the general
topography of thalamocortical projections is normal. We placed DiI crystals in
three locations of the neocortex at E16.5
(Crandall and Caviness, 1984;
Molnar et al., 1998a)
(Fig. 2). In wild-type embryos,
a DiI injection in the occipital neocortex labeled thalamic cell bodies and
cortical axons in the dLGN (Fig.
2A,B,D,E). A DiI injection in the parietal neocortex, however,
labeled cells and axon terminals in a more medial thalamic domain, where the
presumptive ventrobasal complex (VB) is located
(Jones, 1985)
(Fig. 2G,H). Finally, a DiI
injection in the frontal neocortex labeled cells and axons in an even more
medial domain, which includes the presumptive ventromedial nucleus (VM)
(Jones, 1985)
(Fig. 2K,L). In
Ebf1-/- mutant embryos, DiI injections in these three
neocortical zones systematically labeled cell bodies and axons in a thalamic
domain located more medially than in controls
(Fig. 2A-M). Thus,
thalamocortical and corticothalamic projections are shifted. This medial shift
was confirmed by a double injection in the parietal and occipital neocortex
with DiA and DiI, respectively (Fig.
2N-P). Taken together, in Fbf1-/- embryos,
axons of the dLGN do not reach the occipital neocortex and the topography of
thalamocortical projections is shifted with a given thalamic nucleus
projecting towards a more caudal neocortical domain (Figs
2,
10).
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No apparent defects in the dorsal thalamus and neocortex of
Ebf1-/- embryos
How could such a shift in the topographic organization of projections
occur? Ebf1 is expressed in layer I of the neocortex between E10.5
and E12.5, in the mantle of the dorsal thalamus between E12.5 and E14.5, and
in several nuclei in the embryonic basal ganglia
(Wang and Reed, 1993;
Garel et al., 1997;
Garel et al., 1999)
(Fig. 3). Thus, a
straightforward explanation would be that Ebf1 inactivation perturbs
the positioning or specification of the various thalamic nuclei and/or affects
regionalization within the neocortex.
|
Although the expression pattern of Ebf1 does not suggest a role in
neocortical regionalization, we nevertheless checked whether its inactivation
affected patterning or lamination of the neocortex, based on gene expression
patterns. At E12.5, COUP-TFI, Emx2 and Fgfr3 are expressed
in gradients along the anteroposterior axis of the neocortex
(Simeone et al., 1992;
Liu et al., 2000;
Ragsdale and Grove, 2001;
Muzio et al., 2002) and
participate in early cortical regionalization
(Bishop et al., 2000;
Mallamaci et al., 2000b;
Zhou et al., 2001). Later in
development, at E17.5, the expression pattern of COUP-TFI, Id2, Cdh6,
Cdh8, Epha5 and Epha7 are in gradients or restricted to
presumptive cortical areas and show specific laminar patterns
(Suzuki et al., 1997;
Donoghue and Rakic, 1999;
Mackarehtschian et al., 1999;
Miyashita-Lin et al., 1999;
Nakagawa et al., 1999;
Rubenstein et al., 1999;
Liu et al., 2000). At both
ages, we did not observe any changes in the cortical expression patterns of
these genes in Ebf1-/- mutant embryos
(Fig. 4; data not shown).
|
We next studied the morphology of the dorsal thalamus as well as the
expression patterns of Cdh6, Cdh8, COUP-TFI, Epha4, Epha7, ephrin A2,
ephrin A5, Gbx2, Lhx9 and Sema6a. The expression of these
genes is restricted to specific domains or nuclei in the dorsal thalamus,
allowing us to establish the position and molecular properties of the
developing thalamic nuclei (Suzuki et al.,
1997; Zhou et al.,
1997; Feldheim et al.,
1998; Miyashita-Lin et al.,
1999; Retaux et al.,
1999; Liu et al.,
2000; Nakagawa and O'Leary,
2001) (Fig. 5). In
Ebf1-/- mutant embryos, we did not observe changes in the
expression patterns of these genes between E14.5 and E16.5
(Fig. 5; data not shown).
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Overall, our gene expression analysis shows that Ebf1 inactivation does not severely perturb the molecular regionalization of the neocortex and dorsal thalamus. Thus, the shift in thalamocortical projections is unlikely to result from a general change in the positioning or molecular identity of the different thalamic nuclei.
Ebf1 inactivation affects early thalamic axon pathfinding in
the basal ganglia
To better understand this phenotype, we examined how it develops, by
performing DiI injections in the neocortex and dorsal thalamus in early
embryos, before thalamic and cortical axons meet in the internal capsule.
Using DiI injections in the neocortex at E13.5 and E14.5, we did not detect
any defects in the growth and trajectory of cortical axons into the mantle
zone of the ganglionic eminences, the basal ganglia primordium, in
Ebf1-/- embryos
(Miller et al., 1993;
Metin and Godement, 1996;
Molnar et al., 1998a;
Auladell et al., 2000)
(Fig. 6A-D). However, DiI
injections in the dorsal thalamus at E13.5 showed that, in
Ebf1-/- embryos, the thalamic axons growing into the
ganglionic eminences do not make a sharp turn in direction of the neocortex
(Fig. 6E,F). Instead, they are
misrouted towards the pial surface (Fig.
6E,F). At E14.5 and E15.5, the misrouted thalamic axons begin to
navigate towards the amygdala (Fig.
6G,H, Fig. 1A,B).
Thus, whereas early cortical axons show normal navigation before encountering
thalamic axons, thalamic axons are already misrouted when they enter the basal
ganglia (Miller et al., 1993;
Metin and Godement, 1996;
Molnar et al., 1998a;
Auladell et al., 2000).
|
We examined the different cell populations that have been proposed to guide
thalamic axons in the ganglionic eminences. In wild-type embryos, thalamic DiI
injections retrogradely label cells of the perireticular nucleus, which are
located in the internal capsule and form transient projections with the
thalamus (Mitrofanis and Guillery,
1993) (data not shown). In Ebf1-/-, thalamic
DiI injections label a group of basal ganglia cells that is located where the
internal capsule axonal bundle would normally be in E13.5 embryos (arrow in
Fig. 6F), indicating that at
least some perireticular nucleus cells are normally positioned. Next, we
examined the expression of axonal guidance molecules, such as Sema6a
and netrin 1, which are implicated in regulating the growth of thalamic axons
into and through the basal ganglia. Sema6a encodes a transmembrane
semaphorin (Zhou et al., 1997)
and its inactivation affects the pathfinding of a subset of thalamic axons in
the caudal region of the basal ganglia
(Leighton et al., 2001).
Sema6a is expressed in the basal ganglia and in the dorsal thalamus
(Zhou et al., 1997;
Leighton et al., 2001)
(Fig. 6I,K). In
Ebf1-/- mutant embryos, Sema6a expression in the
thalamus was unaffected at E13.5 and E16.5
(Fig. 6I-L). By contrast,
Sema6a expression was greatly reduced in a group of cells located in
the basal ganglia (Fig. 6I-L).
These cells were located close to the entrance point of thalamic axons in the
basal ganglia (Fig.
3A'-C'), where thalamic axons start to show
pathfinding defects in Ebf1 mutants (compare
Fig. 6E-H with
Fig. 6I-L). On the contrary,
expression domains of the gene for netrin 1, which encodes a secreted molecule
involved in the guidance of thalamic and cortical axons
(Metin et al., 1997;
Richards et al., 1997;
Tuttle et al., 1999;
Braisted et al., 2000), were
not severely modified by Ebf1 inactivation
(Fig. 6M-P).
Overall, in Ebf1 mutants, early thalamic axons fail to normally turn towards the cerebral cortex as they enter the basal ganglia, creating an abnormal tract in the amygdalar region. This defect may be due to a change in molecular properties in specific subsets of basal ganglia cells.
Thalamic axons are caudally shifted in the internal capsule of
Ebf1-/- embryos
Thalamic and cortical axons are topographically ordered along their
trajectory in the internal capsule within the basal ganglia
(Molnar et al., 1998a). We
thus examined if the abnormal turn of thalamic axons inside the basal ganglia
of Ebf1-/- embryos affected the topographic organization
of axons inside the internal capsule.
In E16.5 wild-type embryos, double DiI and DiA injections in the frontal and parietal neocortex (Fig. 7A,B), or in the occipital and parietal neocortex (Fig. 7D,E), show that the axons originating from and projecting to a given neocortical domain form ordered bundles in the internal capsule (Fig. 7B,E). Ebf1 mutants showed a normal organization of these axon bundles (Fig. 7A-F).
|
We next examined the position of axons inside the internal capsule by double DiI and DiA injections into the putative dLGN nucleus and VB complex, respectively. In wild-type embryos, the bundle of axons labeled by dLGN injections runs through the caudal part of the striatum and caudal regions of the neocortex (Fig. 7G,H,J). Conversely, axons labeled by VB injections grow at an intermediate anteroposterior position and invade the parietal neocortex (Fig. 7G,H,J). In Ebf1-/- embryos, dLGN axons form the abnormal tract that travels towards the amygdalar region (Fig. 7I). VB injections label a bundle of axons located inside the internal capsule; however, these are located more caudally, where dLGN-labeled axons would normally be in wild-type embryos (Fig. 7J,K).
Thus, altogether, these results indicate cortical axons originating from a given domain have a normal navigation and topography in the internal capsule in Ebf1-/- embryos. Conversely, thalamic axons show a shift in their position inside the internal capsule, and, consistent with their new trajectory invade a more caudal region of the neocortex. Thus, we observe a global shift in the position of thalamic axons just after their turn into the basal ganglia.
Dlx1/2 inactivation affects the topography of
thalamocortical projections
Our analysis of the Ebf1 phenotype indicates that a misguidance of
specific thalamic axons and a general shift in the position of the others can
occur in the absence of apparent defects in the neocortex and dorsal thalamus.
As Ebf1 is expressed in the dorsal thalamus, the possibility that its
inactivation may affect thalamic neurons cannot be excluded. Nevertheless, our
results raise the possibility that developmental defects in the basal ganglia
of Ebf1-/- embryos may shift the topography of
thalamocortical connections. To further examine if affecting structures on the
path of thalamic axons can deviate the topography of thalamocortical
projections, we examined Dlx1/2 -/- mutant embryos
(Qiu et al., 1997).
Dlx1 and Dlx2 encode homeodomain transcription factors and
are expressed in the basal ganglia and in the ventral thalamus, but not in
neocortical projection neurons or in the dorsal thalamus
(Bulfone et al., 1993).
Dlx1/2-/- mutant embryos have a block in differentiation
of the basal ganglia (Anderson et al.,
1997). DiI injections in the neocortex and dorsal thalamus of E14
Dlx1/2-/- embryos shows that both cortical and thalamic
axons fail to make a sharp turn into ganglionic eminences and are displaced
towards the pial surface (Fig.
8A-F). This defect is probably due to the expansion of the
subventricular zone (SVZ*) in the basal ganglia
(Anderson et al., 1997) and
results in the formation of a displaced and highly disorganized internal
capsule, as shown by DiI injections in the dorsal thalamus of E16.5 embryos
(Fig. 8G-J). Although some
thalamic axons reach the neocortex, a large number of the misrouted axons
remain in the basal ganglia (Fig.
8G-J). The identity of the thalamic nuclei generating the axons
that fail to reach to cortex could not be unequivocally determined because of
the disorganization of the internal capsule.
|
The topography of thalamocortical projections was examined by introducing DiI and DiA crystals in the occipital or parietal neocortex of E16.5 heterozygous and homozygous embryos (Fig. 9A-L). These experiments retrogradely labeled fewer cells and axons in homozygous embryos than in controls (Fig. 9A-L), confirming that numerous thalamic axons do not reach the cortex. However, DiI injections (Fig. 9A-I) or double DiI and DiA injections (Fig. 9J-L) in the neocortex of Dlx1/2-/- embryos systematically labeled cells located in a more medial domain (Fig. 9A-F) or in a wider domain that extends more medially (Fig. 9G-L) than in controls. Thus, the topography of thalamic projections into the neocortex is systematically shifted in Dlx1/2-/- mutants.
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We next examined the positioning of thalamic axons within the internal capsule at E16.5 by placing DiI and DiA crystals inside the presumptive dLGN and VB (Fig. 9M-Q). In Dlx1/2-/- embryos, dLGN axons are present in ventral parts of the internal capsule (Fig. 9N,O) but most of these axons do not grow dorsally into the neocortex (Fig. 9P,Q). VB axons are shifted to a more caudal position within the internal capsule, particularly in its dorsal parts (Fig. 9P,Q). Thus, in Dlx1/2-/- mutants, a majority of dLGN axons fail to reach to neocortex and the topography of the remaining thalamocortical projections is shifted in the internal capsule and neocortex, as in the Ebf1-/- mutants.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thalamic projections are shifted in the absence of apparent thalamic
and neocortical defects in Ebf1 and Dlx1/2 mutant
embryos
We show that Ebf1 inactivation drastically affects the pathfinding
of a subset of thalamic axons and creates a shift in the topography of the
remaining thalamocortical projections
(Fig. 10). This shift is
observed in the absence of apparent thalamic or neocortical defects between
E13.5 and E17 (Figs 4,
5) and is first detected within
the basal ganglia primordium (Fig.
10). Furthermore, during the period when thalamic axons are
traveling through the basal ganglia primordium (E13.5-E15.5)
(Miller et al., 1993;
Metin and Godement, 1996;
Molnar et al., 1998a;
Auladell et al., 2000), this
structure exhibits molecular defects
(Garel et al., 1999),
including an abnormal expression of Sema6a
(Fig. 6I-L). Thus, although the
loss of Ebf1 expression in the dorsal thalamus may affect thalamic
neurons, our molecular analysis shows that the shift in topography is not due
to a general change in the molecular regionalization of the neocortex or
dorsal thalamus. Furthermore, our results raise the possibility that this
shift may be due to defects in the basal ganglia.
If defects in structures located on the path of thalamic axons can shift
the topography of thalamocortical axons, we would expect to observe a similar
phenotype in other mutant mice that have basal ganglia defects. We thus
examined Dlx1/2 mutants where differentiation of the basal ganglia
and ventral thalamus is abnormal (Anderson
et al., 1997; Marin et al.,
2000; Yun et al.,
2002). Furthermore, Dlx1 and Dlx2 are not
expressed in the dorsal thalamus or in cortical projection neurons
(Bulfone et al., 1993;
Stuhmer et al., 2002). In
Dlx1/2 mutants, the formation of the internal capsule is perturbed,
probably because of a block in basal ganglia differentiation
(Anderson et al., 1997;
Yun et al., 2002) and, as in
Ebf1 mutants, the topography of thalamocortical projections is
shifted in the neocortex and internal capsule (Figs
9,
10). Thus, the phenotype of
Dlx1/2-/- mice supports the possibility that affecting
structures on the path of thalamic axons can shift the topography of
thalamocortical projections. It could be argued that the reduction of cortical
interneurons in Dlx1/2 mutants
(Anderson et al., 1997) might
contribute to this phenotype. However, this is unlikely because
Nkx2.1 mutants, which also have a major deficit in neocortical
interneurons, have normal thalamocortical projections
(Marin et al., 2002).
Thus, our combined study of Ebf1 and Dlx1/2 mutants shows that the topography of thalamocortical axons can be systematically shifted in the absence of apparent abnormalities in the neocortex and dorsal thalamus, and suggests that this shift is due to defects in structures located on the path of the axons.
The specificity of thalamic axons targeting is not dictated by cues
located within the neocortex
Specific aspects of the phenotypes of Ebf1 and Dlx1/2
mutant embryos provide information on the mechanisms controlling the initial
topography of thalamocortical projections. In particular, the systematic and
coherent shift in the topography of thalamocortical axons indicates that
projections of a given thalamic nucleus are not established independently of
the projections of the other nuclei. In addition, this shift suggests that the
mechanisms involved are likely to control the relative rather than absolute
position of axons. This has been observed in the retinotectal system
(Brown et al., 2000),
suggesting that this feature may be a common characteristic for the formation
of topographic projections.
Another aspect of the phenotypes of Dlx1/2 and Ebf1
mutants is that thalamic axons have a shifted position inside the basal
ganglia before entering aberrant regions of the neocortex. If gradients of
guidance cues within the neocortex were strictly governing the organization of
thalamic inputs, we would have expected the thalamic axons to be redirected to
their appropriate destinations within the neocortex. Thus, positional
information within the neocortex cannot correct the ectopic trajectory of the
thalamic projections, at least at the early developmental stages we have
studied. Therefore, either positional cues do not normally have a central role
in regulating early thalamocortical projections, or perturbing axons on their
way to the neocortex over-rides the activity of instructive gradients. These
observations are in agreement with in vitro experiments in which thalamic
axons can invade any region of the neocortex
(Molnar and Blakemore, 1991).
Furthermore, these data suggest that the initial organization of
thalamocortical projections is not strictly regulated by a `classical'
chemoaffinity mechanism, where axons are guided by positional cues within the
target structure (Drescher et al.,
1997; Feldheim et al.,
2000; Brown et al.,
2000).
The internal capsule: a decision point for the topography of
thalamocortical projections?
Our results suggest that the positioning of thalamic axons inside the
internal capsule is important for the topography of the initial projection, as
changes in this position correlate with changes in the neocortical target
domains. What mechanism(s) could underlie this process? There is evidence that
thalamic axons use cortical subplate axons as a scaffold to enter the cortex
(McConnell et al., 1989;
Ghosh et al., 1990;
Ghosh and Shatz, 1992;
Ghosh and Shatz, 1993;
Molnar et al., 1998b). The
handshake model proposed that thalamic and cortical axons interact inside the
internal capsule and guide each other to their final target, potentially
through specific molecular interactions or through positional alignment within
the internal capsule (Molnar and
Blakemore, 1995; Molnar et
al., 1998a). Neocortical patterning is likely to regulate the
molecular properties and navigation trajectories of cortical axons that grow
towards the thalamus. For example, both Emx2 and COUP-TFI
mutant mice, which show caudal-to-rostral changes in the molecular properties
and connectivity of the occipital neocortex
(Bishop et al., 2000;
Mallamaci et al., 2000b;
Zhou et al., 2001), also have
subplate defects (Zhou et al.,
1999; Mallamaci et al.,
2000a) that may contribute to the changes in thalamocortical
connectivity.
In Ebf1 mutants, we did not observe defects in the pattern or timing of subplate or cortical plate axonal outgrowth into the internal capsule (Fig. 6A-D). However, thalamic axons show pathfinding defects as they enter the basal ganglia, suggesting that the phenotype we observe may be due to a mismatch between normally positioned cortical axons and shifted thalamic axons. In Dlx1/2 mutants, neocortical axons are displaced in the basal ganglia, and thus probably contribute to the disorganization of the internal capsule and to the reduction in the number of axons reaching their target structures (Fig. 8). However, the timing of their outgrowth, and their rostrocaudal distribution, does not seem affected. Thalamic axons, however, had a shifted position within the internal capsule (Fig. 9). Therefore, while defects in both thalamic and cortical axons could contribute to the shift in thalamocortical projections, our observations suggest that it may be primarily due to the displacement of the thalamic axons. Thus, if thalamic and cortical axons do directly interact (`handshake'), our data suggest that a displacement in thalamic axons, and in the alignment of thalamic and cortical axons, can induce a shift in their final target zone in the neocortex as well as a reciprocal shift in cortical projections. Overall, our study suggests that the position of thalamic axons in the basal ganglia, an intermediate structure located between the thalamus and neocortex, is an important decision point for the initial topography of thalamocortical projections.
The role of basal ganglia in guidance and positioning of thalamic
axons
Previous work has identified groups of cells in the basal ganglia that
participate in the guidance of thalamic and cortical axons, by producing
chemoattractants (Metin and Godement,
1996; Metin et al.,
1997; Richards et al.,
1997; Braisted et al.,
1999; Braisted et al.,
2000) and or by forming transient axonal scaffolds
(Mitrofanis and Guillery,
1993). Defects in some of these cells, caused by the
Mash1 mutation, are correlated with the inability of thalamocortical
axons to reach the neocortex (Tuttle et
al., 1999). These results support the idea that the basal ganglia
form an intermediate target for axons interconnecting the neocortex and
thalamus (Metin and Godement,
1996). Our results provide evidence that the ordering of thalamic
axons within the basal ganglia may play an important role in the final
topographic organization of thalamocortical projections.
How is this order established or maintained? Although our study does not
provide definitive answers, it allows us to discuss different hypotheses. One
possibility is that an array of guidance cues regulates the spatial
organization of thalamic axons inside the basal ganglia, by a chemoaffinity
mechanism within this structure. However, so far, there is no obvious
candidate molecule for such function. An alternative mechanism involves both
guidance cues and the timing of thalamic axon outgrowth
(Molnar and Blakemore, 1995;
Molnar et al., 1998a). Indeed,
there is a gradient of differentiation in the dorsal thalamus
(Jones, 1985) and thalamic
axons of different zones grow into the internal capsule at different times
(Molnar et al., 1998a).
Localized guidance cues or groups of cells may determine the position of
pioneering thalamic axons. Candidate molecules include Sema6a: a subpopulation
of thalamic axons is misrouted in the amygdalar region of Sema6a
mutants (Leighton et al.,
2001). These pioneer axons would provide a landmark for the next
set of incoming axons, which would navigate to an adjacent location. Thus,, as
new axons arrive, they would stack in a temporally determined array. In both
models, the shift in topography we observe in Dlx1/2 and
Ebf1 mutant embryos could be a consequence of the axonal pathfinding
defects of specific thalamic axons within the basal ganglia. This mechanism
may also participate in the phenotype of Emx2 and COUP-TFI
mutant embryos, where a subset of thalamic axons do not reach the neocortex
(Zhou et al., 1999;
Mallamaci et al., 2000b;
Lopez-Bendito et al.,
2002).
Conclusion
The favored model for the formation of topographic projections proposes
that this process is regulated by cues located in the two interconnected
structures (Sperry, 1963).
This mechanism is implicated in the targeting of retinotectal
(Drescher et al., 1997;
Goodhill and Richards, 1999;
Feldheim et al., 2000) and
reticulogeniculate (Feldheim et al.,
1998) axons, of limbic thalamic axons to the limbic cortex
(Barbe and Levitt, 1992;
Mann et al., 1998) and
hippocampal neurons to the septum (Gao et
al., 1996), as well as in the formation of a topographic map
within a neocortical area (Vanderhaeghen
et al., 2000). We have presented evidence that the initial
topography of thalamocortical projections is not strictly determined by the
information located inside the target structure and that the position of axons
within intermediate structures may be important for the regulation of this
topography. In particular, our results suggest a role for the basal ganglia in
the organization of thalamic axons and the choice of their final target
destination. More generally, these observations raise the possibility that
intermediate structures, and/or the relative position of axons inside a fiber
pathway, may regulate the formation of topographically organized long-range
projections within the central nervous system.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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